Background and Significance Nanoparticles (particles < 100 nm) have generated increasing interest in the field of medicine as tools for disease detection, and drug delivery. The field of nanomedicine, while still in its infancy, holds tremendous promise as we begin to understand issues related to the benefits, toxicity and environmental impact of nanoscale materials. Colloidal gold is a neutral gold particle synthesized through the combination of gold chloride and sodium citrate. It has been used safely for decades as a therapeutic for patients with arthritis. The particle measures 20-30 nm in diameter and can be linked irreversibly to proteins, peptides, synthetic drugs and nucleotides. In addition to its properties as a nano-carrier, colloidal gold also acts as a high-Z element. Theoretically, the irradiation of high-Z elements at their K-edge absorption energy leads to emission of Auger electrons and photoelectrons, releasing a large amount of energy at their immediate vicinity. This secondary radiation will result in extra damage to tumor cells at the same dose of the applied external radiation. We believe that this secondary property of colloidal gold opens a significant area of novel research and clinical application. Specifically, because of the preferential trafficking of the CYT-6091 particles to tumor tissue, we can utilize this property to enhance the efficacy of external beam irradiation by increasing the radiation dose deposited into the tumor while minimizing toxicity to normal tissues. The fact that CYT-6091 is currently being evaluated as single agent therapy in the context of a Phase I clinical trial within the CCR, makes the potential for the translation of our findings combining CYT-6091 with radiation in the laboratory to the clinic significant. Research Design Aim 1. In order to identify irradiation conditions optimal for production of secondary radiation in gold particles and to estimate the increase of the radiation dose due to the presence of gold nanoparticles, radiation transport calculations will be carried out using the ESG4 Monte Carlo simulation package or the treatment planning software developed within the ROB Radiation Physics Section. The changes of the radiation dose deposited in tissues as a function of the concentration of gold and the quality of the applied external irradiation will be calculated using standard methodology This data will allow us to determine the relative benefit of the addition of colloidal gold as well as the best concentrations of colloidal gold and the optimal energy of radiation to be used in order to optimize the enhanced radiation effects. Aim 2. Response of tumor cells to combination of pegylated colloidal gold TNF nanoparticles with various types of radiation will be characterized by the clonogenic survival assay. Human breast cancer cell lines, MCF7 and MDA251, with different sensitivities to TNF, will be incubated with or without gold nanoparticles and TNF, and exposed to different doses (0 8 Gy) of radiation. The energy of applied x-rays will vary in the range from 5 keV to 60 MeV. The effects of radiation on cell survival will be quantified and evaluated. This series of experiments will help in the validation of the predicted doses from Aim 1 and will allow for the selection of the appropriate cell line, and radiation type and dose for in vivo studies. Aim 3. Initially, CT scans of a tissue-equivalent phantom with inserts containing different concentrations of gold will be used to assess the changes of density of tissue due to the presence of gold nanoparticles and the feasibility of using CT scans to monitor their in vivo biodistribution. Results of the CT scans will be compared to the quantification of the particles in the tissue by validated electron microscopy approaches. If the results of these studies are positive, the micro-CT scanner will be used to compare the changes of Hounsfield units on the CT scans with gold concentration measured in the tissue. Mice with measurable subcutaneous tumors (approximately 0.5 cm3) will be injected with CYT-6091 at doses that may lead to concentrations in the tumors identified as effective by the computer simulations and the in vitro studies. Three and five hours post injection, CT scans will be carried out. Groups of five mice per time point will be used. After completion of the scans, the mice will be euthanized and tissues of interest including tumor, muscle, lung, liver, spleen, pancreas, kidney, stomach, skin, and bone will be collected. All mouse studies will be performed under approved IACUC protocols. The concentration of gold in the tissues will be measured using mass spectrometry techniques. Electron microscopy will be used to assess the sub-cellular microdistribution of gold. Aim 4. The possible synergistic effect of combining x-rays with CYT-6091 will be studied in vivo using both mouse xenograft and syngeneic tumor models. Groups of 10 tumor-bearing animals will be treated with: 1) radiation only; 2) pegylated colloidal gold TNF only; and 3) combination radiation plus pegylated colloidal gold TNF. Non-treated animals will be used as controls. Applied doses of the nanoparticles and radiation will be identified in previous Aims. Tumor size and survival will be used to assess the efficacy of each treatment. Project Status: 1. In vitro clonogenic survival experiments have been carried out to establish the effect of nanoparticles in combination with radiation on cell survival. 2. Preliminary in vivo studies using tumor bearing mice are carried out